Maximum likelihood estimation for cytogenetic dose-response curves.

In vitro dose-response curves are used to describe the relation between chromosome aberrations and radiation dose for human lymphocytes. The lymphocytes are exposed to low-LET radiation, and the resulting dicentric chromosome aberrations follow the Poisson distribution. The expected yield depends on both the magnitude and the temporal distribution of the dose. A general dose-response model that describes this relation has been presented by Kellerer and Rossi (1972, Current Topics on Radiation Research Quarterly 8, 85-158; 1978, Radiation Research 75, 471-488) using the theory of dual radiation action. Two special cases of practical interest are split-dose and continuous exposure experiments, and the resulting dose-time-response models are intrinsically nonlinear in the parameters. A general-purpose maximum likelihood estimation procedure is described, and estimation for the nonlinear models is illustrated with numerical examples from both experimental designs. Poisson regression analysis is used for estimation, hypothesis testing, and regression diagnostics. Results are discussed in the context of exposure assessment procedures for both acute and chronic human radiation exposure.

Is cancer of the colon familial in cotton-top tamarins?

Ten cases of spontaneous adenocarcinoma of the colon were observed in first-generation Saguinus oedipus oedipus born and raised under defined colony conditions. In this series of 10 cases, 3 animals had a wild parent (caught and imported) that also died of cancer of the colon and 4 of the remaining 7 were two unrelated sets of siblings (one a set of fraternal twins). This somewhat unexpected familial association or clustering of these malignancies suggests the possibility of a significant hereditary component to the development of cancer in these tamarins. The histopathology of the tumors in S. o. oedipus is compatible with a genetic influence, as it is strikingly similar to a dominantly inherited mucoid adenocarcinoma of the colon in humans.

Evaluations of cellular proliferation and chromosome breakages after in vitro exposure of human lymphocytes to calcium or zinc DTPA.

The analysis of mitotic indices (MI) and chromosome breakages in metaphases of 50-hr lymphocyte cultures exposed to the calcium or zinc chelates of diethylenetriamine pentaacetic acid (DTPA) demonstrated: (1) an 80% reduction in MI in cultures from three women but no reduction in those from two men after in vitro exposure to CaDTPA in concentrations as low as 10 micrograms/ml culture medium, and complete suppression of mitoses in cultures from men and women after exposure to 40 micrograms/ml CaDTPA; (2) minor suppression in MI in cultures from women and none in those from men after exposure to 40 or 80 micrograms/ml ZnDTPA; (3) no ring or dicentric chromosomes in 1700 metaphases from DTPA-treated cultures. Likewise, in other experiments we observed no differences in the frequency or distributions of rings and dicentrics in lymphocyte cultures from two persons after in vitro exposure to 250-R 60Co gamma radiation in the presence or absence of 10 micrograms/ml CaDTPA or 10 or 80 micrograms/ml ZnDTPA. These data indicate that while accurate estimates of the frequencies of radiation-induced rings and dicentrics in lymphocytes can be made in actinide-contaminated persons undergoing DTPA chelation therapy, blood samples for cytogenetic cultures should not be obtained from chelated patients until the compound has been cleared from the blood plasma.

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes. III. A postimplantation analysis of streptonigrin effects.

The frequency of consistent structural chromosome aberrations was determined for 108 fetuses (day 14 of gestation) from 20 female rabbits treated before conception with 90 micrograms/kg of streptonigrin (NSC-45383). The findings by karyotype analysis of 1.85% of the fetuses with consistent aberrations was compared with 6.32% frequency previously observed in 6-day (preimplantation) blastocysts from identically treated females and found to be significantly reduced. The interpretation of the findings is that the developmental events of implantation and placentation are effective in eliminating from further gestation the majority of the rabbit conceptuses with consistent structural chromosome abnormalities. The data are also discussed relative to the predictive nature of somatic cell chromosome damage in women of childbearing age being an estimator of risk of producing offspring with structural chromosome aberrations. The data show that preconceptional treatment of female rabbits with streptonigrin caused a shift in the cytogenetically determined sex ratio of the 14-day fetuses, but not of the 6-day blastocysts. The significant decrease in relative numbers of males observed is consistent with the induction of lethal mutations on the X chromosome. This finding is discussed in conjunction with the timing and parental specificity of X chromosome inactivation during mammalian embryonic development.

In vivo SCE analysis using bromodeoxyuridine, iododeoxyuridine, and chlorodeoxyuridine.

Sister-chromatid exchange (SCE) frequencies were determined for mouse and rabbit bone-marrow cells following incorporation of 3 different halogenated analogues of thymidine. For both species the SCE frequency was highest for chlorodeoxyuridine, lowest for iododeoxyuridine and intermediate for bromodeoxyuridine. The study demonstrates that halogenated pyrimidine analogues other than brominated compounds can be used for in vivo SCE analysis and that their effects on baseline SCE frequencies are qualitatively similar to results of previous in vitro studies.

Probing sister chromatid exchange formation with halogenated pyrimidines.

This communication describes the use of 6 different halogenated pyrimidine analogues, bromodeoxyuridine (BrdUrd), chlorodeoxyuridine (CldUrd), iododeoxyuridine (IdUrd), bromodeoxycytidine (BrdCyd), chlorodeoxycytidine (CldCyd), and iododeoxycytidine (IdCyd), to achieve sister chromatid differentiation (SCD) and evaluate sister chromatid exchange (SCE) formation in mitogen-stimulated human lymphocytes. Also included are a description of an in vivo experiment with BrdUrd, CldUrd, and IdUrd; a discussion of pyrimidine metabolism effects on SCEs; and the presentation of an update on the "conformation hypothesis" for SCE formation. This hypothesis revision includes a model that centers on the idea that the sum of the conformational alterations of the DNA polymerase-DNA template complex at replication is the controlling factor in SCE formation.

Persistence of SCE-inducing lesions after G0 exposure of human lymphocytes to differing classes of DNA-damaging chemicals.

We conducted studies to determine whether cycling human lymphocytes are equally efficient in repairing sister chromatid exchange (SCE)-producing lesions induced by differing classes of DNA-damaging chemicals. Lymphocytes were pulse-treated during G0 with mitomycin C (MMC), N,N',N''-triethylenethiophosphoramide (ThioTEPA), ethylmethanesulfonate (EMS), or cis-diamminedichloroplatinum (cis-DDP). Bromodeoxyuridine (BrdUrd) was added to the 72 hr cultures at 0 hr or at 48 hr after phytohemmagglutinin stimulation. The concentrations of chemicals employed induced a greater than 2-fold increase in SCEs in second-division metaphases from lymphocytes cultured in the presence of BrdUrd for the entire 72 hr. The analysis of SCEs in uniformly harlequinized metaphases from G0-treated lymphocytes cultured in BrdUrd for the terminal 24 hr showed no increase above baseline after exposure to MMC, and intermediate increases above baseline after exposures to ThioTEPA and cis-DDP. However, after G0 treatment with EMS, the observed SCE frequency was consistent with that expected had all DNA lesions persisted and continued to give rise to SCEs during 3 cell cycles. These findings suggest that cycling human lymphocytes are not equally efficient in eliminating SCE-producing lesions after exposure to differing classes of DNA-damaging chemicals.

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C. Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions.

We conducted a series of experiments designed to determine whether DNA damage induced in G0 lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3-4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G0 lymphocytes are probably expressed as SCEs during the first period of mitogen-induced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.

Sister chromatid exchange distributions in rabbit lymphocytes treated with streptonigrin.

Streptonigrin (NSC-45383), a direct-acting clastogen which induces SCEs in vivo and chromosome aberrations both in vivo and in vitro, was evaluated for SCE induction in both G0 and stimulated rabbit lymphocytes. Determinations were made for 16 cultures from seven female rabbits. These included controls as well as cells exposed to 90 micrograms/kg in vivo, cells pulse-treated with 50 ng/ml in vitro, and a culture continuously exposed to 5 ng/ml in vitro. For all cultures the SCE/cell frequency was determined from 20 complete (44 chromosome) metaphases and, in selected cultures, SCEs on individual chromosomes (880 per culture from 20 cells) were enumerated to determine SCE/chromosome frequency and the chromosomal distribution of SCEs. Analysis of variance and least significant difference tests of the square root x transformed SCE/cell data show that cells exposed to streptonigrin while dividing have significantly higher (P less than 0.01) frequencies (over double the control 5.3 SCE/cell value) whereas treated G0 cells were not significantly different from the controls. Dispersion analysis of both SCE/cell and SCE/chromosome data confirms the adequacy of the Poisson distribution for spontaneous or baseline but not streptonigrin-induced SCEs.

SCE frequencies in rabbit lymphocytes as a function of time after an acute dose of cyclophosphamide.

Following acute and chronic exposures to various chemicals in vivo, the average SCE frequency in human and rabbit lymphocytes has generally been shown to decrease with time posttreatment. The rate of this decline varies, however, and little data have been published pertaining to the decrease in SCEs soon after exposure. To gain more information about the immediate decline in SCEs with time, we injected rabbits with a single dose of 35 mg/kg cyclophosphamide (CP) and determined SCE levels in circulating lymphocytes at various times 5 h to 2 weeks after treatment. We observed a rapid decline in SCE frequencies within 5 days, and by 10 days post-exposure the SCE levels were back to control values. The distribution of SCEs among cells and the number of circulating lymphocytes were also analyzed at each time. Within 2-3 days posttreatment we observed a rapid loss of cells with high SCE levels concomitantly with a rapid decline in circulating lymphocytes and a decrease in the average SCE frequency. When the number of lymphocytes began to increase, the number of cells with normal SCE values also increased. By 10-11 days after CP, the lymphocyte count had recovered, the SCE frequency had returned to control levels, and the distribution of SCEs among cells was almost identical to the control distribution. These data, in addition to published information on rabbit lymphocyte lifespan, suggest that the decline in SCE levels with time posttreatment is a function of lymphocyte turnover.

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes. I. The test system and the effects of streptonigrin.

An appropriate method for evaluating transmissible genetic damage in female germ cells is presented. Analysis of karyotype preparations from preimplantation (6- day) rabbit embryos for consistent structural chromosome abnormalities is described as a sensitive way in which these determinations can be made. A table prepared from summarized research reports shows that less than 1 in 2000 rabbit blastocysts karyotyped thus far has a consistent structural chromosome abnormality. In the new data presented in this study, none of the 90 control blastocysts karyotyped had consistent structural chromosome abnormalities while 13 of 278 from female rabbits treated with streptonigrin (NSC-45383) before conception did. These findings demonstrate that karyotype analysis for consistent chromosome lesions in preimplantation rabbit embryos from females exposed to chemical clastogens can serve as a sensitive and quantitative means of estimating effects on oocytes. The results of the study also show that the consistent structural chromosome lesions in the blastocyst karyotypes are what would be predicted from the mode of action of streptonigrin and the segregation of chromosomes during meiosis. The relative efficiency of this system based on the rabbit as compared to another recently described system bases on the hamster is also discussed.

Physical, chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture.

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G0 exposure in the pH range 6.6-7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4 degrees C vs. 37 degrees C. In other studies, SCE induction was evaluated in cultures exposed during G0 to MMC concentrations ranging from 0.25 to 2.5 microgram/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G0 exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G0 exposure time (SCE/microgram/h). A mean frequency of 20.7 +/- 4.8 SCE/microgram/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G0 exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.

Analysis of mutagen-induced chromosome damage in a primate species (Saguinus oedipus oedipus) at risk for spontaneous adenocarcinoma of the colon.

A high incidence of adenocarcinoma of the colon (greater than 16%) has been observed at necropsy in the South American primate, Saguinus oedipus oedipus (S. oedipus), while the disease has not been found in tamarins of the closely related species, Saguinus fuscicollis spp, housed in the same research colony. Cytogenetic analyses in cultured lymphocytes from 10 S. oedipus and 10 S. fuscicollis illigeri (S. fuscicollis) demonstrated no differences in the average frequencies of spontaneous or mitomycin C (MMC)-induced sister chromatid exchanges (SCEs) between animals of the two species. However, highly significant variability in MMC-induced chromosome lesions was observed between the individual S. oedipus, with one animal exhibiting increased sensitivity for both SCEs and chromosome breakages. At present we do not know the relationship, if any, between increased sensitivity to mutagen-induced cytogenetic lesions in specific S. oedipus tamarins and the increased risk for colon cancer that has been documented in this primate species. However, our cytogenetic findings in this one S. oedipus are similar to data obtained in evaluations of persons with several autosomal recessive conditions in which there is a genetic predisposition for developing malignancies.

Factors affecting the sensitivity of rabbit lymphoid and bone marrow cells to Streptonigrin-induced chromosome aberrations.

In previous cytogenetic studies of rabbits injected with the clastogen, streptonigrin (SN), we observed a higher incidence of chromosome aberrations in lymph node cells than in marrow cells recovered 6 h after treatment. In this study we examined several factors that may be responsible for this difference in the incidence of lesions. Results from SN pulse-treated lymphoid and marrow cultures demonstrated that the responses were different in vitro also. In addition, at higher doses of SN, lymphoid cells displayed a greater sensitivity to the drug when treated during DNA synthesis; marrow cells showed no differences in sensitivity throughout the cell cycle. Analysis of the percentage of lymph node and marrow metaphases labeled following 2, 4, or 6 h of culture in the presence of tritiated thymidine indicated that G2 was less than 2 h in lymph node cells and approximately 4 hr in marrow cells. From these data we conclude that the difference in the incidence of lesions 6 hr after an in vivo SN exposure. However, our data also indicate that lymph node cells are innately more sensitive to the induction of lesions than are marrow cells.

Cytogenetic effects of cis-platinum(II)diamminedichloride in vivo.

The chemotherapeutic agent cis-platinum(II)diamminedichloride (cis-PDD) has been shown to be mutagenic, teratogenic, and carcinogenic. We determined the cytogenetic effects of cis-PDD on human and rabbit lymphocytes in vitro and on rabbit marrow cells, lymph node cells, and lymphocytes in vivo. Lymphocyte cultures from two humans and one rabbit were treated in vitro with cis-PDD. For in vivo studies, five New Zealand white rabbits were given iv injections of cis-PDD. Posttreatment blood samples were withdrawn for analysis and rabbits were sacrificed at either 6 or 24 hr for cytogenetic analysis of marrow and node cells. Sister chromatid exchange (SCE) analysis of human and rabbit metaphases from lymphocytes treated in vitro showed that rabbit lymphocytes are more sensitive to SCE induction by cis-PDD. Significant increases in SCE were observed in lymphocyte cultures obtained as early as 1 hr post treatment from injected rabbits. Analysis of node, marrow, and lymphocyte metaphases from injected rabbits showed a high number of chromosome aberrations in these cells with bone marrow showing a delayed response to treatment. These results indicate that cis-PDD is clastogenic in hematopoietic tissues in vivo and that SCE methodology may be useful in monitoring patients receiving cis-PDD therapy.

Chromosomal radiation sensitivity in ataxia telangiectasia long-term lymphoblastoid cell lines.

Elevated frequencies of spontaneous chromosome breakage and hypersensitivity to radiation induced chromosome damage are characteristic findings in cultured lymphocytes and skin fibroblasts of patients with ataxia telangiectasia (AT). To determine whether long-term AT lymphoblastoid cells (B-lymphocytes) which do not express spontaneous chromosomal instability, exhibit increased chromosomal radiation sensitivity, four AT lymphoblastoid cell lines were exposed to various doses of 250 kVp X-rays during G2. At doses of 25 and 50 rad, the AT cells exhibited a 2-3 fold increase in radiation damage relative to that observed in B-cell lines or PHA stimulated lymphocytes from normal donors, while at 100 rad, the AT cells demonstrated approximately 1.5 times more lesions. These findings suggest that the genetic (or other) factors responsible for increased chromosomal radiation sensitivity in AT lymphocytes and fibroblasts also operate in long-term lymphoblastoid AT cells.

The effect of washing lymphocytes after in vivo treatment with streptonigrin on the yield of chromosome and chromatid aberrations in blood cultures.

Cytogenetic analyses of cultured blood lymphocytes from streptonigrin-treated female rabbits demonstrated that the yield of chemically-induced chromatid aberrations, expecially exchanges, is influenced by the method of blood handling before in vitro culture. When blood cells from treated animals were washed with tissue-culture medium before culture, chromatid breaks were observed at the same level as in the controls, and no chromatid exchanges were found. However, when duplicate aliquots of blood were inoculated directly into culture medium, without washing, elevated levels of chromatid breaks were observed and chromatid exchanges were found. These findings indicate that the clastogen, streptonigrin, was carried into the culture medium with the blood-cell inoculum.